PROJECT SUMMARY Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle wasting disease caused by dystrophin deficiency, which leads to respiratory, orthopedic, and cardiac complications. DMD predominantly affects males, leading to an average lifespan of 19 years with approximately 20% of deaths due to cardiac failure. Currently, there is no cure for DMD. The mdx mouse model of DMD captures the orthopedic and respiratory aspects of the disease but does not accurately recapitulate the cardiomyopathy that is observed in humans. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from DMD patients are available but have so far only been assessed in two-dimensional configurations. There are currently no 3D human in vitro cardiac tissue- engineering strategies available to accurately model physiological cardiac function. Without accurate functional disease models of DMD cardiomyopathy, the mechanistic pathways that contribute to progressive decline in cardiac function cannot be elucidated. To this end, we propose to engineer a 3D biomimetic cardiac ventricular model (VM) using hiPSC-CMs and cell-sheet engineering to investigate the effects of tissue architecture on pumping function within the dystrophic heart. Patients with DMD often lack the functional dystrophin protein in cardiac muscle, but do not present with cardiomyopathy until later in life. This suggests that there is a compensatory mechanism present in young dystrophic tissues. The myocardium is well understood to have anisotropic muscle layers organized in a helical pattern where myofibrils within each layer changes from +60 to -60 from the endocardium to the epicardium, respectively. Taken together, we hypothesize that the intrinsic helical structure of the myocardium improves ejection fraction and compensates for the weaker contraction forces of dystrophic CMs. To test this hypothesis, we will utilize hiPSC-CMs derived from healthy and DMD patients and nanopatterned cell-sheet stacking technology to create tissue-engineered VMs with layer-by-layer control over cellular orientation. Structure-function relationships within VMs will be evaluated through three modalities: (1) pressure generation through intraluminal catheterization, (2) cardiac output via echocardiography, and (3) electrophysiological endpoints. Both healthy and dystrophic VMs with helical architectures are expected to generate more pressure than VMs with unrepresentative architectures. This approach will provide the most biomimetic human in vitro model of the heart to date and the first human engineered tissue model of DMD cardiomyopathy capable of producing physiological functional output. This platform could be utilized as a more advanced preclinical screening tool for potential therapeutic drug compounds and their effects on human heart function.